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Repression of Retrotransposal Elements in Mouse EmbryonicStem Cells Is Primarily Mediated by a DNAMethylation-independent Mechanism*□S

Received for publication, March 23, 2010, and in revised form, April 16, 2010 Published, JBC Papers in Press, April 19, 2010, DOI 10.1074/jbc.M110.125674

Leah K. Hutnick‡§, Xinhua Huang‡, Tao-Chuan Loo‡, Zhicheng Ma‡, and Guoping Fan‡1

From the ‡Department of Human Genetics and §Neuroscience Interdepartmental Program, David Geffen School of Medicine,UCLA, Los Angeles, California 90095

In defense of deleterious retrotransposition of intracisternalA particle (IAP) elements, IAP loci are heavily methylated andsilenced in mouse somatic cells. To determine whether IAP isalso repressed in pluripotent stem cells by DNA methylation,we examined IAP expression in demethylatedmouse embryonicstem cells (mESCs) and epiblast-derived stem cells. Surpris-ingly, in demethylated ESC cultures carryingmutations of DNAmethyltransferase I (Dnmt1), no IAP transcripts and proteinsare detectable in undifferentiated Oct4� ESCs. In contrast,�3.6% of IAP-positive cells are detected in Oct4� Dnmt1�/�

cells, suggesting that the previously observed increase in IAPtranscripts in the population of Dnmt1�/� ESCs could beaccounted for by this subset of Oct4� Dnmt1�/� ESCs under-going spontaneous differentiation. Consistent with this pos-sibility, a dramatic increase of IAP mRNA (>100-fold) andprotein expression was observed in Dnmt1�/� ESC culturesupon induction of differentiation through the withdrawal ofleukemia-inhibitory factor for 6 or more days. Interestingly,both mRNAs and proteins of IAP can be readily detected indemethylatedOct4� epiblast-derived stem cells as well as dif-ferentiatedmouse embryo fibroblasts, neurons, and glia uponconditional Dnmt1 gene deletion. These data suggest thatmESCs are a unique stem cell type possessing a DNA methy-lation-independent IAP repression mechanism. This methy-lation-independent mechanism does not involve Dicer-medi-ated action of microRNAs or RNA interference because IAPexpression remains repressed in Dnmt1�/�; Dicer�/� doublemutant ESCs. We suggest that mESCs possess a unique DNAmethylation-independent mechanism to silence retrotrans-posons to safeguard genome stability while undergoing rapidcell proliferation for self-renewal.

DNA methylation, an epigenetic modification where a methylgroup is covalently added to the cytosine of CpG dinucleoti-des, is catalyzed by a family of DNA methyltransferases

(Dnmts)2 that include de novo (Dnmt3a and -3b) and main-tenance methyltransferases (Dnmt1) (1, 2). DNA methyla-tion is known to regulate developmental gene expression,genomic imprinting, X-inactivation, and genomic stability(3–6). Many retrotransposable repetitive elements are heav-ily methylated, and current evidence supports a causal rela-tionship between DNA methylation and repression of retro-transposons (7–9). Intracisternal A particles (IAPs) aremurine endogenous retroviral repetitive elements, with anestimation of over 1000 copies across the haploid murinegenome (10–12). Germ line mutations due to IAP retro-transposition most often occur at intronic sites, disruptinggene expression through premature termination, aberrantsplicing, or viral LTR-driven transcription (13). Further-more, insertional mutagenesis of IAP elements is noted invarious murine cancer cell lines with subsequent activationof oncogenes or cytokine genes (14, 15).When expressed, IAP proteins are non-infectious viral

particles that are retained in the cisternae of the endoplas-mic reticulum (16). IAP proteins can be transiently detectedin preimplantation embryos (17, 18), although DNAmethyl-ation of IAP elements is largely maintained throughoutembryogenesis (19, 20). In established lines of mouse embry-onic stem cells (ESCs) derived from the inner cell mass, IAPelements are already heavily methylated with little detecta-ble IAP expression (21).Here, we examine expression of IAP proteins in control

and demethylated somatic cells, EpiSCs, and mESCs. Usinga previously generated antibody against IAP protein (p73)(22) as well as our polyclonal antibody raised against re-combinant IAP gag protein (9, 23), we mapped changes inIAP protein levels after Dnmt1 gene deletion in floxed(Dnmt12lox/2lox) mouse embryonic fibroblasts (MEFs) usingvirus-mediated cre recombinase. Unexpectedly, we foundthat IAP mRNA and protein levels are not detected in Oct4�

Dnmt1�/� ESCs but are dramatically increased in demethy-lated mESC cultures upon in vitro differentiation, suggestiveof the presence of DNA methylation-independent repres-

* This work was supported, in whole or in part, by National Institutes of HealthGrant RO1 NS051411 and P01 GM081621 (to G. F.). This work was also sup-ported by California Institute of Regenerative Medicine Grant RC1-0111 (toG. F.) and National Research Service Award 5F31NS051 (to L. K. H.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 A Carol Moss Spivak Scholar in Neuroscience. To whom correspondenceshould be addressed: Dept. of Human Genetics, David Geffen School ofMedicine, UCLA, 695 Charles Young Dr. S., Los Angeles, CA 90095. Tel.:310-267-0439; Fax: 310-794-5446; E-mail: [email protected].

2 The abbreviations used are: Dnmt, DNA methyltransferase; IAP, intracister-nal A particle; ESC, embryonic stem cell; mESC, mouse ESC; EpiSC, epiblast-derived stem cell; MEF, mouse embryo fibroblast; GST, glutathione S-trans-ferase; LIF, leukemia-inhibitory factor; RT, reverse transcription; FISH,fluorescent in situ hybridization; PBS, phosphate-buffered saline; LTR, longterminal repeat; MSCV, murine stem cell virus; GFP, green fluorescent pro-tein; Cre, cre recombinase; En, embryonic day n.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 27, pp. 21082–21091, July 2, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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sion mechanism(s) in silencing IAP expression in undiffer-entiated mESCs.

EXPERIMENTAL PROCEDURES

Antigen Generation and Anti-IAP2 Purification—Sequencefrom plasmid M1A14 (23) was used to clone the IAP2 frag-ment (amino acids 250–750) into pGEX4T3 vector (Fig.1) (9). After isopropyl 1-thio-�-D-galactopyranoside induc-tion, bacteria were harvested, and the IAP2-GST fusion pro-tein was purified with glutathione beads (Amersham Bio-sciences). After evaluation of the thrombin-cut eluate, antigenwas shipped to PickCell Laboratories for rabbit immunoinjec-tion. Rabbit polyclonal serum was tested and evaluatedbefore purification. Briefly, GST-IAP2 fusion protein wasadsorbed onto glutathione beads (Roche Applied Science),fixed by 20 mM dimethyl pimelimidate in 200 mM HEPESbuffer, pH 8.5, and terminated in 200 mM Tris-HCl buffer,pH 8.3. Antibody bound to IAP2-GST fusion protein waswashed with Tris-buffered saline and eluted by 200 mM gly-cine (pH 2) followed by neutralization with 1 M Tris-HCl, pH8.5, buffer to pH 7.4. Eluates were examined by immunoblot-ting at different dilutions in three independent trials to verifyspecificity.Viral Production and Infection—MSCV-Cre-GFP and MSCV-

GFP were generated in HEK 293T cells via calcium phosphatetransfection. For viral infection, cells were trypsinized andresuspended in 1 ml of cell medium (90% Dulbecco’s modifiedEagle’s medium, 10% fetal bovine serum, 1� penicillin/strep-tomycin, 1 mM glutamine) with Polybrene (8 �g/ml). Cellsin suspension were incubated with titered virus. After 30min, the infected cells were added to gelatinized wells andcoverslips. Medium was changed after 6–8 h. During thetime course, corresponding wells were harvested for DNA,RNA, and protein. Coverslips were retrieved and fixed in 4%paraformaldehyde for 15 min at room temperature forimmunocytochemistry.Lentivirus (LV-Cre-GFP) was generated by the UCLA Depart-

ment of Medicine VectorCore with a concentration of 1.6 �107 plaque-forming units/ml. cre recombinase activity wasverified by infecting fibroblasts generated from the R26R�geo line (24). To infect mESCs, cells were grown feeder-freefor two passages, and after trypsinization, 5 � 105 cells wereresuspended in 1 ml of LV-Cre supernatant plus 8 �g/mlPolybrene and rotated in Eppendorf tubes for 2 h at 37 °C.Cells were pelleted, washed twice with medium, and platedat low density onto feeders with coverslips to monitor acuteinfection. For subcloning analysis, infected single cells wereenriched using flow cytometry sorting (FACsVantage, UCLAJCCC Flow Cytometry Core) and plated at low density, andindividual colonies were picked and expanded for DNA,RNA, and immunohistochemistry.Epiblast Stem Cell Derivation and Culture—Dnmt12lox/2lox

mice were mated, and at E5.5, postimplantation embryoswere dissected and cultured as described with minor modi-fications to the protocol (25, 26). EpiSC cultures were main-tained on �-irradiated feeders in EpiSC medium (Dulbecco’smodified Eagle’s medium/F-12 supplemented with 7 �g/mlinsulin, 15 �g/ml transferrin, 5 mg/ml bovine serum albu-

min, 12 ng/ml FGF-2, 20 ng/ml activin A, 1000 units/ml LIF,1� penicillin/streptomycin, and 450 �M monothioglycerol).EpiSCs were passaged using collagenase dispase dissociationinto small colonies. For lentiviral infection, EpiSCs were dis-sociated in 0.25% trypsin-EDTA, preplated to remove feed-ers, and infected using the above described procedure.Mouse ESC Culture—Wild type control (J1 cells),Dnmt1�/�

(c/c) ESCs (27), Dnmt[1(kd), 3a�/�, 3b�/�] (TKO cells (28), akind gift from Dr. Rudolf Jaenisch, Whitehead Institute), andEed�/� mESCs (generously provided by Dr. Terry Magnuson,UNC) were cultured under standard conditions on DR4 �-irra-diated feeders, changing medium daily (85% high glucose Dul-becco’s modified Eagle’s medium, 15% ESC screened fetalbovine serum (Hyclone), 1 mM glutamine, 1� penicillin/strep-tomycin, 0.1 mM non-essential amino acid, 0.1 mM �-mercap-toethanol, 500 units LIF/ml) and passaging using trypsin-EDTA. For partial differentiation, mouse ESCs were platedon 0.2% gelatin B-coated plates and coverslips. After 24 h(Day 0 time point), medium was changed to LIF� differentia-tion medium (90% Dulbecco’s modified Eagle’s medium, 10%fetal bovine serum, 1� penicillin/streptomycin, 1 mM gluta-mine). Medium was changed every day, with correspondingwells harvested for DNA, RNA, protein, and coverslips over thetime course (Days 0–12).Immunoblotting—The immunoblotting procedure was per-

formed as described previously (29). The original IAP antibody(anti-p73, clone 37.11X from Dr. Kira Lueders) was used at adilution of 1:1000 and incubated overnight at room tempera-ture. Our IAP2 antibodywas used at a 1:2000 dilution forWest-ern blots. Anti-glyceraldehyde-3-phosphate dehydrogenase(Abcam; 1:5000) was used as loading control.RNA Purification and Quantitative RT-PCR—Total RNA

was harvested by TRIzol as per the manufacturer’s instruc-tions (Invitrogen). RNA was converted to cDNA using theiScript reaction kit (Bio-Rad). Quantitative RT-PCR was per-formed using Bio-Rad MyCycler and SYBR Green Supermix(Bio-Rad). Results were normalized to 18 S values expressed as-fold change relative to corresponding control values (30). pvalues were assessed using Student’s t test (two-tailed, paired).Forward and reverse primers were as follows: IAP forward,AAG CCC TTT TGT TCC TTT TCA; IAP reverse, ACC CTTGGAAAGGCC TGTAT; 18 S forward, GCC CTGTAATTGGAA TGA GTC CAC TT; 18 S reverse, CTC CCC AAG ATCCAA CTA CGA GCT TT.IAP2 Fluorescent in Situ Hybridization (FISH) and North-

ern Analysis—IAP2 (amino acids 250–700) sequence wasPCR-amplified from the MIA14 plasmid gel purified (Pro-mega). 100 ng of IAP2 template was used to generate a Cy3-labeled cDNA probe using the Bioprime kit (Roche AppliedScience) as per the manufacturer’s instructions. FISH wascarried out as previously described (31). The same amplifiedregion was used to create a Northern probe. Northern blotanalysis was performed using the standard procedure in ourprevious publication (29).Immunostaining—Coverslips were picked and fixed in 4%

paraformaldehyde for 15 min and washed three times (5 mineach) with 1� PBS. Blocking and permeabilization were car-ried out for 1 h with 0.5% Triton X-100 in PBS plus 10%

Retrotransposon IAP Regulation in mESCs and EpiSCs

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normal goat serum. Primary antibody diluted in PBS plus 1%normal goat serum and 0.1% Triton X-100 was added tocoverslips and incubated overnight at room temperature.Primary antibodies used were the following: anti-IAP2 (rab-bit polyclonal, 1:1000) and anti-Oct 3/4 (Santa Cruz Biotech-nology, Inc.; mouse monoclonal, 1:20). Anti-H4K20 3me(Millipore; mouse monoclonal, 1:500) was incubated atroom temperature for 1 h. Coverslips were washed, and sec-ondary antibody (Jackson Immunoresearch) diluted in PBSplus 1% normal goat serum and 0.1% Triton X-100 was addedfor 1 h.Section immunohistochemistry was performed as described

previously (32). The original anti-IAP (p73) antibody wasdiluted (1:500) in PBS containing 5% normal goat serum, 3%bovine serum albumin, and 0.25% Triton X-100 overnight atroom temperature. Our IAP2 antibody was used at 1:1000 inPBS plus 5% normal goat serum and 0.25% Triton X. The nextday, the sections were washed in 1� PBS three times for 10mineach, and then secondary antibody (Jackson Immunoresearch)was applied for 2 h at room temperature.Confocal Fluorescence—Images were taken at �63 magnifi-

cation using Leica confocal software on a Leica TCS-SP MPConfocal and Multiphoton Inverted Microscope (Heidelberg,Germany) equipped with an argon laser (488-nm blue excita-tion, JDS Uniphase), a 561-nm (green) diode laser (DPSS,Melles Griot), a 633-nm (red) helium-neon laser, and a two-photon laser setup consisting of a Spectra-Physics Millenia X532-nm green diode pump laser and a Tsunami Ti-Sapphirepicosecond pulsed infrared laser tuned at 768 nm for UVexcitation.

RESULTS

IAP Antiserum Detects the gagProtein Encoded in IAP Elements,Which Is Expressed in DemethylatedFibroblast Cells—Given that IAPrepeats are heavily methylated byDnmt1, we asked whether IAPprotein could be detected in DNAdemethylation models. The origi-nal p73 antiserum also recognizesother proteins containing partialproducts of IAP coding regions(23) (Fig. 1B). For specific recogni-tion of IAP gag protein product, wecreated a recombinant proteinfusing GST to a partial fragment ofIAP gag protein (IAP2, amino acids251–699) (Fig. 1A). Once purifiedover IAP2-GST columns, the origi-nal serumyieldedacleanp73bandviaimmunoblot in tissue possessing over95% Dnmt1�/� cells in the centralnervous system (32) (Fig. 1B). Thepurified serum was partially blockedby preadsorption against IAP2 pep-tide fragment, indicating the specific-ity of antiserum(datanot shown).We

FIGURE 1. IAP protein is only detected in lysates of E18.5 Dnmt1�/�

mutant brain. A, depiction of protein structural domains of the full-lengthIAP clone MIA14. A deletion series of the gag peptide domain, which mostclosely corresponds to the p73 peptide used to generate the original IAPantibody (45), was cloned to create GST fusion peptides. B, immunoblottingrevealing IAP expression in control (Con) and E18.5 Nestin-Cre; Dnmt1 condi-tional mutant brain lysate (Mut) of the original p73 serum before purification(anti-p73, left blot) and after column purification using the IAP2-GST fusionpeptide (anti-p73, middle blot). Our IAP2 polyclonal antibody also stronglyrecognizes the 73-kDa gag protein (anti-IAP2, right blot) from mutant brainlysate (32). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 2. IAP protein reactivation is tightly linked to the onset of DNA demethylation in MEFs. A, Dnmt12lox/2lox MEFs were infected with MSCV-Cre, and genomic demethylation was monitored using restrictionenzyme digestion with methyl-sensitive HpaII followed be Southern blot analysis of IAP repetitive elements.Genomic demethylation appears 4 days after infection (DIV). B, immunoblotting shows that IAP protein expres-sion appears 5 days postinfection with MSCV-Cre; thus, the IAP protein expression occurs after significantgenome demethylation. The far right lane is a positive control for IAP proteins detected in the brain of E18.5Nestin-Cre; Dnmt1 conditional knockouts (Brain E18.5 MUT) (29). C, IAP immunochemistry (red) colocalizes withMSCV-Cre-GFP-infected cells (green) once the genome becomes significantly demethylated after DNMT1 dele-tion. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4�,6-diamidino-2-phenylindole.



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used this recombinant IAP2 protein fragment to generate a sepa-rate polyclonal antibody for both immunoblotting and immuno-staining assays to detect IAP protein expression (Fig. 1B).

To examine the onset of IAP pro-tein reactivation after Dnmt1 genedeletion, Dnmt12lox/2lox MEFs wereinfected with retrovirus containingcre recombinase fused to green fluo-rescent protein (MSCV-Cre-GFP)(33). Significant genomic demethy-lation was detected after 4 dayspostinfection via Southern blottingfor the IAP repeat probe (Fig. 2A).Because DNA methylation levelsdropped after the deletion ofDnmt1, we found reactivation ofIAP protein translation in infectedcells starting 5 days postinfectionvia immunoblot (Fig. 2B). Whenindividual cells were examined forIAP immunoreactivity, IAP proteinwas detectable in a minority ofinfected cells as early as 4 dayspostinfection (Fig. 2C). IAP expres-sion was restricted to the Cre-GFP-infected cell population (Fig. 2C,arrows). Thus, the reactivation ofIAP protein expression is tightlyassociated with the onset of genomicDNA demethylation caused by thedeletion ofDnmt1.IAP Protein Immunostaining

Marks Demethylated Cells and Cul-tured Neuroblastoma Cells at a Sin-gle Cell Resolution—We next askedwhether IAP protein could be usedto detect demethylated cells at asingle cell resolution in the devel-oping nervous system after condi-tional Dnmt1 deletion in neuralprecursors. Using our anti-IAP2, IAPimmunoreactivity was restricted tothe zone of Dnmt1 deletion as dic-tated by the expression pattern ofEmx1-Cre and Nestin-Cre (Fig. 3, Aand B) (9, 32). In the Emx1-Cre-driven deletion of Dnmt1, no im-munoreactivity for IAP was seen incontrol regions of striatum, thala-mus, brain stem, or cerebellum,where DNA methylation is main-tained (data not shown).The neuroblastoma cell line N2a,

which is known to transcribe certainIAP-LTR-containing genes (34, 35),shows strong anti-IAP2 immunore-activity in the cytoplasm with acharacteristic juxtanuclear staining

pattern for viral A particles (Fig. 3C). Endogenous IAP proteinlevels are strongly reactivated in N2a cultured cells, which cor-relates with the known hypomethylation of IAP elements in the

FIGURE 3. Detection of IAP protein expression in demethylated somatic cells. A, left, immunoblotting usinganti-IAP2 shows IAP protein in control and E18.5 Nestin-Cre; Dnmt1 conditional mutant brain lysates. Right, IAPimmunoreactivity in E18.5 dorsal cortex of E18.5 Nestin-Cre; Dnmt1 mutant mice (29). B, left, Western blotanalysis of IAP protein expression in control and Emx1-Cre; Dnmt1 cortical lysate from neonate P0 to 1.5 yearsold. Right, IAP immunoreactivity in the dorsal cortex (ctx) of 6-month-old Emx1-Cre; Dnmt1 mutant mice (9).C, immunocytochemistry for IAP protein in cultured N2a neuroblastoma cell lines. Western blot showing IAPexpression in N2a cells but not in other types of neuroblastoma cells, including B104 and Hs 683 from ATCC.GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4�,6-diamidino-2-phenylindole.



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N2a genome (35). Thus, IAP immunoreactivity is a very usefultool to locate demethylated cells in a variety of DNA demethy-lation models.Demethylated Embryonic Stem Cells Do Not Contain Detect-

able IAPmRNAs and Proteins yet Exhibit Dramatic IAP Induc-tion upon Differentiation—Dnmt1�/� mESCs possess less that22% normal genomic methylation levels and exhibit increasedexpression of IAP mRNAs (27, 28, 36). Surprisingly, when weperformed Western blot analysis to assay IAP proteins, wecould not detect IAP proteins in lysate from undifferentiatedDnmt1�/�mESCcultures (Fig. 4A).Whenweused IAPFISH todetect mRNA signal in the undifferentiated Dnmt�/� mESCs,wedid not visualize increased IAPmRNA levels (Fig. 4E,Day0).We thenperformedco-immunostainingof IAPandOct3/4 tover-ify whether individual cells express IAP protein in the undifferen-tiated state. Only a small minority of IAP-positive cells existed inDnmt1�/� cultures (3.6 � 0.51%, mean � S.E., n� 825 cells; Fig.4D,Day 0), but all IAP positive cells were Oct3/4-negative, indic-ative of differentiation. IAPprotein reactivation in thisminorpop-ulationmay be below immunoblot detection levels, yet the level ofIAP transcripts was elevated sufficiently in this differentiated sub-set to account for detection by more sensitive quantitative RT-PCR assay in the population ofDnmt1�/� ESCs.We next examined the time course of IAP immunoreactivity

in demethylated mESCs after in vitro differentiation. Upon LIFwithdrawal in the absence of feeder cells, Dnmt1�/� mESCs

undergo a dramatic increase in IAPtranscription and translation (Fig. 4,B and C). Quantitative RT-PCR re-veals a 10-fold increase in IAP tran-script levels 5 days after LIF with-drawal inDnmt1�/� cells. Moreover,a similar profile of mRNA expres-sion is detected using FISH labelingand Northern blot analysis specificfor IAP2 (Fig. 4, E and F). After 6days of LIF withdrawal, IAP proteinis detectable in total cell lysate (Day6 long exposure; Fig. 4C). By Day 9of the LIF withdrawal time course,we see robust protein expression(Fig. 4C) and a corresponding 103�29-fold relative increase of IAPtranscription in Dnmt1�/� cultures(p� 0.0001; Fig. 4B).Z-stack confo-cal microscopy through individualcolonies revealed IAP immunoreac-

tivity present only in cells with low or no Oct3/4 immunoreac-tivity throughout the time course (Fig. 4D). Over the courseof LIF withdrawal, the differentiated population increasesdramatically (Fig. 4D), and only after a significant increase inthis population was the threshold for immunoblot detectionreached.Detection of IAP Protein Expression in Demethylated EpiSCs—

To determine if the DNA methylation-independent mecha-nism in suppression of IAP elements is unique to pluripotentstem cells, such as mouse ESCs, we derived Dnmt12lox/2lox

EpiSCs and examined the effect of DNA demethylation on IAPexpression. EpiSCs are derived from the epiblast of the post-implantation embryo at E5.5, express pluripotent stem cellmarkers, such as Oct4 and Nanog, and form cells of the threegerm layers in vitro and in vivo (25, 26). However, distinct dif-ferences in morphology, culture conditions, and gene expres-sion profiles set EpiSCs apart from mESCs. Because EpiSCsexhibit up-regulation of endodermal and ectodermal markerswhen compared with mESCs, EpiSCs are regarded as more lin-eage-committed in developmental pathways.Using lentivirus to deliver cre recombinase, we monitored

infectedDnmt12lox/2lox EpiSCs for IAP protein reactivation. By2 days postinfection, IAP protein was dramatically up-regu-lated in Oct3/4-positive cells (Fig. 5). IAP signal remained highin the infected (GFP�) EpiSCs and their derivatives in early

FIGURE 4. Expression of IAP protein is suppressed in Dnmt1�/� mESCs and is only reactivated upon partial differentiation coupled with dramaticincreases in IAP mRNAs. A, IAP protein is not expressed in undifferentiated Dnmt1�/� mESCs, although Dnmt1�/� cells possess less than 22% of normalgenomic DNA methylation levels (28). Scale bar, 25 �m. B, upon induction of differentiation of Dnmt1�/� (c/c) mESCs in the absence of LIF treatment, IAP mRNAlevels increase dramatically over the time course of 6 –9 days in culture. No induction of IAP expression was observed in wild type control (J1) mESCs. C, IAPprotein levels in the heterogeneous lysate of Dnmt1�/� cells were also detected after 6 days of LIF withdrawal, reaching a detectable threshold by Western blot.The blot on the left is a longer exposure to show detectable IAP expression at Day 6 of LIF withdrawal. D, IAP immunocytochemistry shows that onlyOct4-negative cells express IAP protein in Dnmt1�/� ESCs. IAP-expressing cells were detected in a small population (under 10%) at Day 0 – 4 of differentiationbut reached peak levels at Day 6 –9 upon the induction of differentiation by LIF withdrawal. Scale bar, 50 �m. E, IAP FISH analysis reveals that both wild type (J1)and Dnmt1�/� (c/c) undifferentiated colonies at Day 0 of LIF withdrawal do not express IAP mRNA. Upon LIF withdrawal to induce differentiation, partiallydifferentiated Dnmt1�/� cells migrating away from the colony express IAP RNA (arrowheads). Dnmt1�/� cells with undifferentiated colony morphology (arrow)do not express IAP mRNA. Scale bar, 25 �m. F, Northern blot analysis of the IAP gag coding region reveals the dramatic induction of IAP mRNA as LIF iswithdrawn from culture. -Fold induction levels are assessed after normalization of 28 S rRNA bands from wild type (J1) and Dnmt1�/� (c/c) during the timecourse of differentiation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4�,6-diamidino-2-phenylindole. Error bars, S.E.

FIGURE 5. Hypomethylated EpiSCs rapidly induce IAP protein reactivation. A, photomicrographs of arepresentative colony 2 days after lentiviral cre recombinase delivery to Dnmt12lox/2lox EpiSCs. IAP immunore-activity is present in Oct4-positive (blue) infected (GFP-positive, green) EpiSCs. Scale bar, 25 �m. B, 4 days afterlentiviral cre recombinase delivery, colonies arising from infected EpiSCs show a homogenous population ofOct4/IAP/GFP-positive cells. Scale bar, 25 �m.



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passages. However, demethylated EpiSCs do not survive pas-saging3 and behave more like demethylated MEF cells (33).

Examine Potential DNA Methylation-independent IAP Re-pression Mechanism(s) in Demethylated mESCs—Dnmt1�/�

mESCs appear to possess an alternative mechanism unique toembryonic stem cells to silence IAP elements in the absence ofDNA methylation. Inhibition of histone deacetylases shifts chro-

3 L. K. Hutnick, X. Huang, T.-C. Loo, Z. Ma, and G. Fan, unpublishedobservations.

FIGURE 6. Inhibition of histone deacetylases and proteosome activities does not lead to IAP protein expression in undifferentiated Dnmt1�/� mESCs.A, HDAC inhibitor treatments of Dnmt1�/� mESCs in the presence of LIF were performed to question whether blocking histone deacetylation, thus promotingthe active chromatin conformation, in Dnmt1�/� mESCs would lead to IAP protein expression in undifferentiated cells. Confocal images show that HDACinhibitors do not reactivate IAP protein expression in Dnmt1�/� mESCs, suggesting that an alternative repressive mechanism is involved. Scale bar, 25 �m.B, cell counts show that HDAC inhibitors increase the number of IAP-positive colonies, which correlates with the known differentiating effect of HDAC inhibitorsin cell culture. C, after 8 h of treatment with the proteosome inhibitor MG132, IAP protein was not visualized in Oct3/4-expressing Dnmt1�/� mESCs (confocalphotomicrograph; scale bar, 25 �m). Error bars, S.E. DAPI, 4�,6-diamidino-2-phenylindole.



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matin to a permissive state for transcription, relieving chromatinrepression in mESCs(37). Histone deacetylase inhibitors signifi-cantly increased the number of IAP-positive mESC colonies aswell as increasing thenumber of IAP-positive cells per colony (Fig.6). However, IAP-positive cells did not colocalize with strongOct3/4 immunoreactivity (Fig. 6A); thus, cell differentiation, aknownsideeffectof treatmentwithHDACinhibitors, explains theincrease in IAP immunoreactivity.The 20 S proteasome acts as a transcriptional silencer block-

ing nonspecific transcription initiation at intergenic and intra-genic regions in mESCs (38). Because the majority of IAP LTRinsertions are intergenic, we investigated whether this mecha-nism controls unwanted transcription of IAP elements. Aftertreatment with the proteosome inhibitor MG132 for 8 h, nocolocalization of Oct3/4 and IAP immunostaining was seen incolonies examined (Fig. 6C); nor was a dramatic rise of IAPmRNA present in treated Dnmt1�/� mESCs (0.58 � 0.17-foldchange, p � 0.08). Because IAP reactivation was not seen ineither wild type or demethylated mESCs after MG132 treat-

ment, proteosome-mediated degra-dation of intergenic gene transcrip-tion probably is not the alternativemechanism.It has been postulated that Dicer

protein-mediatedRNA interferenceormicroRNAproduction could be apotential mechanism to block IAPexpression. Minor demethylationof IAP elements is detected inDicer�/� mESCs (39). We thereforegenerated Dnmt1�/�; Dicer�/� dou-ble mutant ESCs using lentiviral crerecombinase and examined byimmunocytochemistry for the colo-calization of IAP and Oct4. Individ-ual subclones were genotyped viaPCR for 2lox versus 1lox detectionat both loci, and quantitative RT-PCR verified the reduced expressionof both Dnmt1 and Dicer (Fig. 7).Although IAP protein was stronglyexpressed in spontaneously differen-tiating cells (Oct3/4-negative), wefailed to detect the co-localizationof strong IAP signal with Oct3/4-positive cells (Fig. 7). It is of notethat two clones showed a few cells(�0.2% of the counted colonies)with weak juxtanuclear staining(Fig. 7). Our results argue againstthe possibility that Dicer-mediatedRNA interference or microRNAswould be the alternativemechanismin repressing IAP expression inundifferentiated mESCs.

DISCUSSION

Host cell defensive strategieshave evolved to counteract the deleterious consequences of IAPelement reactivation. The best documented mechanism, DNAmethylation of LTR promoters, can directly impede access oftranscription factors or lead to an inactive form of chromatin attarget loci (40). As the time course of Dnmt1 conditional dele-tion in MEF cells indicates, IAP protein expression is tightlycorrelated with genomic DNA methylation levels, making IAPprotein reactivation a reliable indicator of global changes inDNA methylation levels in individual cells. Previous tools forexamining global DNA hypomethylation levels relied on evalu-ating DNA or RNA extracted from a population of cells.Although these techniques are sensitive, no evaluation could bemade for global DNA methylation changes in individual cellswithin tissue. As shown in Fig. 3, IAP immunohistochemistryallows for recognition of DNA demethylation at a cellular levelin different tissue types. In cultured N2amouse neuroblastomacells, a robust reactivation of IAP protein is present. Similarly,in hypomethylated neural lineage cells, IAP protein can be usedto trace the demethylation status at the individual cell level.

FIGURE 7. Clonal analysis of IAP expression in Dicer�/�; Dnmt1�/� mESCs. A, IAP protein reactivation wascounted in individual clones after genotyping was performed. The vast majority of IAP-positive cells wereOct3/4-negative, thus indicating that only Dicer/Dnmt1 double mutant mESCs express IAP after spontaneousdifferentiation. Of note, two clones had a few cells expressing weak levels of IAP protein in Oct3/4-positive cells.The numbers on the x axis denote the clone number for the 3– 6 individual clones with one of the followinggenotypes: Dnmt12lox; Dicer2lox (control); Dnmt11lox; Dicer2lox (Dnmt1�/� mutant cells); Dnmt11lox; Dicer1lox

(Dnmt1�/�; Dicer�/� double mutant ESCs)). B, photomicrographs of clone 127, where differential levels of IAPare expressed. Strong IAP immunostaining is seen in differentiating (Oct3/4-negative) cells, whereas a weakperinuclear stain can be occasionally seen in few Oct3/4� cells (arrow, bottom). Scale bar, 25 �m.



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Thus, IAP antibodies are valuable reagents to detect demethy-lated cells in the murine system.Hypomethylated mESCs appear to possess an alternative

mechanism for IAP silencing beyond the DNA methylation-mediated pathway. Interestingly, EpiSCs, which are also pluri-potent yet differ from mESCs in both transcriptional networksand epigenetic modifications (25, 26), show strong IAP proteinreactivation after Dnmt1 deletion. Thus, a methylation-inde-pendent repressional mechanism is unique to mESCs. Furtheranalysis of transcriptional and epigenetic profilesmay highlightthis unique repressional mechanism in mESCs.It can be potentially argued that DNA methylation directed

by de novo methyltransferases in Dnmt1�/� mESCs silencesIAP elements. We evaluated IAP protein expression levels inDnmt1/Dnmt3a/Dnmt3b-deficient mESCs (TKO cells) (28).Under standard mESC culture conditions, the IAP-positiveTKO cell population was not Oct4-positive (supplementalFig. 1). Because TKO cells have less than 2% of the normalmethylation levels (28), we argue that mESCs possess an alter-native mechanism in the absence of DNA methylation thatmediates IAP gene silencing.We have considered the involvement of both transcriptional

and translational mechanisms controlling IAP expression indemethylated mESCs. It is possible that redundant repressivemechanisms are present in mESCs that include DNAmethyla-tion and histone modifications, yet HDAC inhibitors did notinduce IAP expression in demethylatedDnmt1�/�mESCs (Fig.6). Furthermore, preliminary results showed that deficiency ofeither PRC2 (eed�/� mESCs) or PRC1 (Bmi�/� brain tissues)alone did not relieve IAP protein repression (data not shown).To definitively ascertain PcG complex involvement, deficiencyof both polycomb complexes and DNA methylation in mESCsmay be needed to trigger IAP protein expression.Small RNA-mediated silencing pathways have been reported

to regulate retrotransposons. Presently, contradictory reportsregarding Dicer involvement in IAP silencing appear in the lit-erature. One group observed increased transcription from cen-tromeric repeats, L1s, and IAPs (39); however, other groupsstate that mammalian DICER and Eif2c2 (Ago2) have no rolesin maintaining genomic methylation in mESCs, with directevidence that the RISC complex does not repress IAP expres-sion post-transcriptionally (41, 42). Our double deletionmodel inmESCs reveals no large induction of IAP protein reac-tivation, arguing that Dicer-mediated transcriptional silencingis not strongly targeting IAP repression. Although we observedweak IAP immunostaining in a few Dnmt1�/�; Dicer�/� cells,this could be due to the fact that Dicer mutation dramaticallyincreases Oct3/4 levels in mESCs (39, 41). The appearance of afew Oct3/4 immunopositive cells weakly co-stained for IAPcould be explained by longer turnover of Oct3/4 protein; thus,cells undergoing early stages of spontaneous differentiationwould retain residual Oct3/4 protein.While this manuscript was in preparation, two recent pub-

lications demonstrated that KAP1/Trim28 repressor proteincoupled with histone lysine 9 methyltransferase KMT1E(ESET/SETDB1) is involved in repressed IAP gene transcrip-tion in mESCs (43, 44). These studies revealed a novel role ofrepressive histone modifications as an alternative transcrip-

tional repressive mechanism independent of DNA methyla-tion. Interestingly, in the absence of all three Dnmts in mESCs,Matsui et al. (44) reported that the KAP-repressive complexremains associated with IAP retrotransposon elements. Fur-thermore, KAP1/Trim28 acts synergistically with DNA meth-ylation to repress IAP transcription (43). However, neither ofthe two groups have examined whether IAP proteins are pres-ent in KAP1�/� mESCs. In our study, we found that IAP genetranscription is only moderately increased when comparedwith differentiated Dnmt1�/� cells. Furthermore, IAP proteinis not detected in demethylated mESCs, consistent with anadditional inhibitory mechanism that blocks IAP expression inundifferentiated mESCs.Based on the findings of this current study, we conclude that

IAP protein expression can be used to detect DNAhypomethy-lation at the cellular level inmurine cells ranging from epiblast-derived stem cells to adult neurons. Both IAP transcription andprotein translation can be detected upon DNA hypomethyla-tion in lineage-committed cells. However, in undifferentiatedmESCs, there exists a compensatory mechanism(s) to repressretrotransposon elements independent of DNA methylation.The need for an alternative mechanism further highlights theimportance of maintaining genomic stability to prevent inser-tional mutations in fast replicating mESCs.

Acknowledgments—We thank Dr. Kira Lueders for the gifts of the p73antibody and the MIA14 plasmid and Thuc Le for editing of themanuscript.

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